The role and mechanism of macrophage activation induced by exosomes from high glucose-treated renal tubular epithelial cells / 中华肾脏病杂志

ABSTRACT

Objective To investigate the role and mechanism of macrophage activation induced by exosomes from high glucose-treated renal tubular epithelial cells.Methods (1) The supernatant of renal tubular epithelial cells which were cultured in normal glucose control group (5.5 mmol/L D-glucose) or high glucose group (30.0 mmol/L D-glucose) for 48 h were collected and ultracentrifuged to harvest exosomes.Exosomes were identified by transmission electron microscope and Western blotting.(2) Exosomes were labeled with the green lipophilic fluorescent dye PKH67 and cultured with THP-1 macrophage to investigate whether HK2-derived exosomes could be internalized by THP-1 macrophage.Observing the morphology microscopically and detecting the chemotaxis function of THP-1 macrophages in Transwell chamber after co-cultured with exosomes.The expression of inducible nitric oxide synthase (iNOS),tumor necrosis factor-α (TNF-α),Interleukin-1β (IL-1β),monocyte chemoattractant protein-1 (MCP-1) in cells and supernatants were separately detected by quantitative Real-time PCR (qRT-PCR) and enzyme linked immunosorbent assay (ELISA),and the expression of p-c-Jun NH2-terminal kinase (p-JNK),mitogen-activated protein kinase p-p38 (p-p38MAPK) and nuclear factor κB p65 (NF-κB p65) in THP-1 macrophages were detected by Western blot.Results (1) Vesicles that harvested by ultracentrifugation ranged in size from 30 nm to 100 nm and expressed exosomal marker CD63,TSG101 but absence of calnexin which is a marker of endoplasmic reticulum,suggesting that the exosomes were not contaminated with cells.(2) Results from laser scanning confocal microscope showed that each group of exosomes can be internalized by THP-1 macrophages.Compared with normal glucose exosomes group,high glucose exosomes had increased the expression of iNOS,TNF-α,IL-1β and MCP-1 in THP-1 macrophages (all P ＜ 0.01),moreover,p-JNK,p-p38 MAPK and NF-κB p65 proteins level also increased significantly (all P ＜ 0.01).Conclusions Exosomes from high glucose-treated HK2 cells can induce THP-1 macrophage activation and functional changes through MAPK/NF-κB pathway.